In vitro and in vivo studies of cytotoxic effects of FeSO4 nanoparticles

Background and aims: Using iron as a food additive usually causes undesirable sensory changes and side effects in humans. In this study, we made iron (Fe) nanoparticles (NPs) and studied the cytotoxicity of FeSO4 bulk and NPs on HT-29 cells and different doses of these particles on rat intestine. Methods: Particle size of nanoscale was achieved by mechanical technique. Iron particles were characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The effect of iron particles with different concentrations (6.25, 3.125, and 1.57 mM/mL) on the colon cell line was performed using the MTT assay at 24, 48, and 72 hours. Apoptosis and necrosis of the cells were assessed using Annexin V-FITC staining and propidium iodide (PI) at 24 h. In an in vivo study, Taftoon bread was produced from fortified wheat flour with FeSO4 bulk and NPs, which are recommended in human diet (9, 18, and 27 mg of elemental iron/kg flour). Wistar rats were fed daily with fortified bread for 21 days and their colon and small intestine were then evaluated histopathologically. Statistical analyses were performed using SPSS 22.0 software by chi-square test. Results: The synthesized FeSO4 NPs were smaller than 100 nm, and they had more adverse effects on the viability of the HT-29 cells compared to the bulkFeSO4 at 72 hours. Flow cytometric study showed that the early apoptosis of cells by the bulk form was more than the NPs, but at the low concentration (1.57 mM/mL), the NPs induced more necrosis than the bulk particles (P = 0.063). The survival rate of cells facing all concentrations of NPs and bulkFeSO4 decreased dose dependently (P = 0.075). In vivo results revealed that there were no pathological changes in rats’ intestinal tissues. Conclusion: The bulk and NPs of iron have adverse effects on the HT-29 cells, but no histopathological changes were seen on rats’ intestinal cells.


Introduction
Despite the comprehensive application of nanoparticles (NPs) in foods, such as improving flavour and texture, increasing shelf life, enhancing dietary value, the small size of these nanoparticles, i.e. less than 100 nm, and the possibility of their attachment to vital body structures and causing toxic effects, stay a source of concern among scientists, as reflected in the research undertaken (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Moreover, NPs' properties can affect their toxicity to biological systems such as cell culture (13)(14)(15). Therefore, determination of a suitable dose of NPs in cytotoxicity assessments can influence their toxic impacts (16). In addition, global investments in nanotechnology research have dramatically increased (17,18) and numerous cytotoxicity studies are still ongoing to address the growing concern over the toxic effects of NPs on human health (15,19). As the most abundant metal in the environment and an important and well-known micronutrient, iron is vital for biochemical reactions in the body and for producing blood cells. Iron deficiency is also globally prevalent as the most widespread nutritional disorder (20). In the event that the oral iron supplied is insufficient to make red blood cells, the body first uses its reserves, including the iron stored in the liver, leading to iron deficiency anemia if the condition is not treated. The improper distribution of iron-enriched foods, the delayed beginning of supplementation, the insufficient daily dose of prescribed iron, and malnutrition are factors that contribute substantially to iron deficiency anemia. Today, iron deficiency anemia is a prevalent and widespread nutritional disorder during pregnancy in most countries, including Iran, and a major risk factor for increasing the maternal and neonatal mortality and the prevalence of low birth weight and premature birth (21)(22)(23). Enriching foods such as bread with iron supplements is an effective and inexpensive strategy for controlling and preventing disorders associated with iron deficiency or iron deficiency compensation; nevertheless, this strategy can cause problems such as changing the taste and color of the food or gastrointestinal tract complications (24)(25)(26). Iron compounds with the highest bioavailability can cause undesirable sensory changes as they are strongly attracted to nutrient components. The optimal iron compound selected for food enrichment should have the highest relative bioavailability, be cost-effective, and does not cause unacceptable sensory changes in taste, color, and texture of the food. Divalent iron sulfate is a compound with the chemical formula of FeSO 4 , which is widely used as a water-soluble iron enrichment agent for enriching flour owing to its cost-effectiveness. FeSO 4 is a generally recognized as safe (GRAS) compound that is recommended for low-extraction flour enrichment. FeSO 4 salt with the highest bioavailability among iron compounds is a peroxide whose ferrous ions are converted into ferric ions during oxidation. Ferric compounds can change the color of food products into brown, and are less absorbed in the intestine due to its insolubility. Its large particles also cause fairly acceptable darkness in the flour and black spots in the bread (27)(28)(29)(30). Given the extensive use of iron as a food additive, the current research aimed to discover data on ways to reduce undesirable sensory modifications and side effects induced by their bulk form by studying the cytotoxicity of nanosized and bulk iron particles to HT-29 cells and the impacts of separate doses of these particles on the intestine of rats.

Synthesis of NPs and Characterization of Particles
A planetary ball mill made by Fritsch Pulverisette 6 was used to physically synthesize 87% pure FeSO 4 as NPs. To prepare these NPs, the bulk FeSO 4 was poured into the machine and milled at 200 rph for 15 hours with a ballto-powder weight ratio of 10:1 (31).
The particle shape, size and mean diameter of NPs was observed by scanning electron microscopy (SEM; KYKY-EM3200, China) and transmission electron microscopy (TEM; Philips CM 10, Eindhoven, The Netherlands).

In vitro evaluation of the toxicity of iron NPs
The human intestinal HT-29 cell line can be a good model for cytotoxicity assessments of different materials for cells (32). The human colon cell (HT-29) line was procured from the National Cell Bank of Pasteur Institute, Tehran, Iran. These cells were grown on RPMI 1640 Medium (Roswell Park Memorial Institute medium, Gibco, USA) along with 100 U/ml of penicillin, 100 μg/mL of streptomycin, and 10% fetal bovine serum (FBS, Gibco, USA), and incubated at 37 º C in an atmosphere containing 5% CO 2 (33). These cells were cultivated as single layers in a flask. The culture medium was substituted three times a week, and the cells were isolated with trypsinase and passaged at 80%-90% confluence. The cells were ultimately counted and cultured in 96-well plates.

Cell Viability Assay
The MTT assay is a quantitative and staining test based on the reduction of the water-soluble yellow salt of 3-(4.5dimethylthiazol-2yl)-2.5 diphenyl tetrazolium bromide and the formation of insoluble dark-blue formazan crystals in water. The MTT reduction occurs only in viable cells by the mitochondrial enzyme of succinate dehydrogenase. The cells viability is therefore measured with the MTT assay. Colon cells (1×10 4 cells/well) were fully grown in 24 hours on the 96-well plates in RPMI 1640 medium. After reaching 80-90% cell confluence, the supernatant was replaced with a new medium containing different doses of nanosized and bulk iron particles, i.e. 6.25, 3.125, and 1.56 mM/mL. The selected doses were based on the previous studies (34,35), and the test was performed with three interactions. After 24, 48, and 72 hours, 10 μL of filter-sterilized MTT (5 mg/mL in PBS, Melford) was added to each well and incubated for 4 hours at 37 º C. In the next step, the Formazan crystals produced were dissolved into each well by adding 100 μl of DMSO (Roth, Germany), and the produced stain was read at 570 nm using an ELISA reader. The viability was calculated using the relative absorbance and expressed as the percentage of that of the control sample (36).
Apoptosis and necrotic cells were evaluated by using Annexin V staining and Propidium iodide (PI). The cells were cultivated for 24 hours with distinct levels (6.25, 3.125, and 1.56 mM/mL) of the FeSO 4 bulk and NPs. Cells were detached and stained with PE-Annexin V and PI, based on the manufacturer's protocol of Annexin V apoptosis detection kit I (BD Pharmingen TM ) and analyzed by flow cytometry (Partec, Germany).

Production of iron-enriched bread
Wheat flour was first obtained from Danesayan Factory™ in Kerman, Iran, and mixed with a certain amount of iron NPs and bulk iron. After adding the yeast (Saccharomyces cerevisiae, PTCC 5080), 55%-60% water and 1%-2% salt, bread containing 9, 18, and 27 mg of iron/kg of flour was prepared (37).

In vivo evaluation of the toxicity of iron NPs
Preparation and grouping of animals A total of 49 Wistar rats (Rattus norvegicus) weighing 280±20 g were procured from the national laboratory animal house in Kerman, Iran. The animals were kept in polyvinyl carbonate cages for one week in an animal house at 22±2 º C and humidity of 55±5% and in 12:12 lightdark cycles (38). They had free access to food and water. All the ethical principles of conducting animal research were observed. The rats were then randomly divided into seven groups (n=7 in each), including a control group. Three groups treated daily with bread enriched with bulk iron containing 9, 18, and 27 mg of iron/kg of flour and, three groups treated daily with bread enriched with iron NPs with the same dose for 21 days (39)(40)(41). These groups were carefully examined for emerging clinical symptoms. After the period of treatment, rats were euthanized using 80 mg/kg ketamine and 2.5 mg/kg Acepromazine IV and histological samples were taken.

Histopathology
Samples ultimately were taken from the colon and small intestine of the rats, and were fixed with 10% formalin. Hematoxylin and eosin (H&E) stain was used to examine the pathological changes.

Statistical analysis
The data were analyzed in SPSS 22 software by chi-square test, and P < 0.05 was considered a significant difference. The data obtained from histopathological studies were also investigated and expressed using descriptive statistics.

Characterization of particles
The particles' morphology was nanospheres, and the mean particle size decreased to less than 100 nm after nanonization ( Figure 1).

Cell proliferation and metabolic activity
The results showed that HT-29 cells metabolic activity in all the three concentration, except for 6.25 mM/mL of bulk particles, decreased after 72 hours in a dose-and time-dependent manner, so that cell viability at different concentrations of bulk FeSO 4 (1.57, 3.125, and 6.25 mM/ mL) was 100%, 96%, and 73.7% at 72 hours, respectively. However, the survival rate of cells facing NPs was 66.9%, 87.4%, and 59.7%. The cell viability decreased by approximately 40.3% of bulk FeSO 4 at high concentration (6.25mM/mL) in the colon cell line.
Furthermore, the results showed that cell viability decreased from 89.2% to 73.7% and from 77.4% to 59.7% by bulk and NPs, respectively, at high concentration (6.25mM/mL) in a time-dependent manner.
The statistical analyses revealed that there were no significant differences among control and treatment groups (P = 0.063). Moreover, no significant difference was seen between groups treated with bulk Fe and Fe NPs (P = 0.75) (Figure 2). Furthermore, flow cytometry determined the amount of necrotic and apoptotic cells. The maximum amount of necrotic cells was associated with a 1.57 mM/mL concentration of bulk FeSO 4 . However, the flow cytometry of the particles found the highest cell viability to be associated with the low concentrations of FeSO 4 NPs, i.e. 1.57 and 3.125 mM/mL (Figures 3 and 4). Therefore, the effect of NPs was more than the bulk particles on the HT-29 cells.

Histopathological Analysis of Rats
The histopathological examination revealed that there were no pathological changes in intestinal cells. Moreover, hemosiderin pigments were not observed in the small intestine and colon tissues of any of the studied samples ( Figure 5).

Discussion
In the present study, FeSO 4 NPs were synthesized using a planetary ball mill, and the cytotoxicity of the NPs and bulk iron particles to HT-29 cells was examined using the MTT assay. The effects of different doses of these particles were also studied on the intestine of the rats fed by bread enriched with these particles. The results showed that the viable cells decreased in groups treated with bulk and NPs of iron, but necrosis and apoptosis in cell lines were more in the bulk iron treatment groups than iron NPs.

Conclusion
The synthesized iron NPs had adverse effects on the HT-29 cancer cells, which intensified by time passage. Contrary to the bulk particles, NPs induced more necrosis in HT-29 cells at the lowest level (1.57mM/mL). Noteworthily, the orally iron particles as a bread fortificant in rats did not provoke histopathological lesions in normal intestinal tissue. Since the iron NPs fortified bread had no detrimental effect on the rats' normal intestinal cells, we suggest to study the interaction of iron with bread constituents at a molecular level.